Methods of treating pigs with Bacillus strains

ABSTRACT

Disclosed are methods of administering at least two  Bacillus  strains to a pig, such as female breeding stock, nursery pigs, or other pigs. The  Bacillus  strains inhibit  Clostridium  in litters borne to the pig. The  Bacillus  strains also are useful when administered to herds lacking symptoms of  Clostridium  infection. Administration of the  Bacillus  strains improves performance of female breeding stock and in piglets borne by the female breeding stock.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 61/036,741, filed Mar. 14, 2008, theentirety of which is incorporated by reference herein.

FIELD DESCRIBED HEREIN

The invention relates to controlling disease in pigs, enhancing pigperformance, and improving the health of pigs with Bacillus strains.

DESCRIPTION OF THE RELATED ART

Enteric clostridial infections in swine occur predominantly in theneonatal period but are also associated with hemorrhagic bowel syndromeaffecting pigs in the finishing period. Although immunization against C.perfringens type C has greatly reduced pre-weaning mortality, nocommercial vaccines are currently available for C. perfringens type A orC. difficile. C. perfringens type A and C. difficile infections are nowrecognized with increasing frequency in neonatal pigs and approaches todiagnosis and prophylaxis are both different and more complex than thosefor type C infections.

There is a lack of efficacious commercial vaccines for C. perfringenstype A and C. difficile. Conventional control strategies for theseclostridial enteric disease include fecal feed-back programs,antibiotics, oregano oil, and probiotics. Unfortunately, the efficacy ofthese therapies has been limited.

Feeding antibiotics such as bacitracin to pigs has many drawbacks tofeeding antibiotics to livestock, including consumer acceptance. Thereis also concern about selection of antibiotic-resistant bacteria.Antibiotics are also expensive and have variable effectiveness. Inaddition, some countries have banned the feeding of antibiotics toanimals. For example, on Jan. 1, 2006, the European Union banned thefeeding of all antibiotics and related drugs to livestock for growthpromotion purposes. The sweeping new policy follows up a 1998 ban on thefeeding of antibiotics that are valuable in human medicine to livestockfor growth promotion. Now, no antibiotics can be used in Europeanlivestock for growth promotion purposes.

Oregano oil has also been used to combat clostridial disease in pigs andimprove health of swine. Etheric oils from oregano exert antibacterialeffects. However, the use of phytogenic feed additives is controversial,and the efficacy of this therapy has been limited.

Pigs and piglets suffer from scours, that is, diarrhea, which can becaused by bacteria such as Escherichia colibacillosis (E. coli) andClostridium perfringens Types A and C. Scours can cause death losses andsevere production losses, including weight loss, if left untreated.

In view of the foregoing, it would be desirable to provide methods ofusing one or more Bacillus strains to treat or prevent Clostridium-baseddisease in pigs and to enhance performance of pigs.

SUMMARY OF THE INVENTION

The invention, which is defined by the claims set out at the end of thisdisclosure, is intended to solve at least some of the problems notedabove. Methods are provided. In one embodiment of the method, aneffective amount of at least two Bacillus strains is administered to apig. The Bacillus strains are selected from strains 22CP1 (ATCCPTA-6508), 3AP4 (ATCC PTA-6506), 15AP4 (ATCC PTA-6507), 2084 (NRRLB-50013), LSSAO1 (NRRL B-50104), and 27 (NRRL B-50105).

In another embodiment of the method, an effective amount of Bacillusstrains 3AP4 (ATCC PTA-6506) and LSSAO1 (NRRL B-50104) is administeredto a pig. The pig can be a gestating pig, a lactating pig, or any otherpig.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred exemplary embodiments described herein are illustrated in theaccompanying drawings, in which like reference numerals represent likeparts throughout and in which:

FIG. 1 is the dendrogram representing the genetic relatedness among 30isolates of C. perfringens recovered from a first set of swabs.

FIG. 2 is the dendrogram representing the genetic relatedness among C.perfringens isolates harvested from a second set of swabs.

FIG. 3 is the dendrogram representing the genetic relatedness among 40isolates of C. perfringens recovered from a third set of swabs.

FIG. 4 is a graph showing clostridia counts (CFU/g of tissue) from theileum and distal colon of pigs from control and DFM supplemented sows(section×treatment interaction: P=0.14, SE=5.5×10⁵; a, b Means without acommon letter differ, P>0.05).

Before explaining embodiments described herein in detail, it is to beunderstood that the invention is not limited in its application to thedetails of construction and the arrangement of the components set forthin the following description or illustrated in the drawings. Theinvention is capable of other embodiments or being practiced or carriedout in various ways. Also, it is to be understood that the phraseologyand terminology employed herein is for the purpose of description andshould not be regarded as limiting.

DETAILED DESCRIPTION

As used herein, “performance” refers to the growth of an animal, such asa pig, measured by one or more of the following parameters: averagedaily gain (ADG), weight, scours, mortality, feed conversion, whichincludes both feed:gain and gain:feed, and feed intake. “An improvementin performance” or “improved performance” as used herein, means animprovement in at least one of the parameters listed under theperformance definition.

The present application provides methods of administering an effectiveamount of one or more Bacillus strains to a pig. In one embodiment, themethods improve performance of pigs. Thus, it may be economical for aswine producer to routinely administer one or more Bacillus strain,either individually or in combination with other Bacillus strains, notonly to treat and prevent disease, but also to improve performance.

In another embodiment administration of one or more Bacillus strainsinhibit pathogenic Clostridium, such as C. perfringens and C. difficilein pigs. The methods may also be used to reduce or even preventclostridial disease in pigs not currently infected with clostridialpathogens.

In at least some embodiments of the method, feeding one or more Bacillusstrain to pigs also include the following: a decrease in percent ofscouring litters of pigs, improved 15 d piglet body weight, improvedpiglet average daily gain (ADG), and decrease in sow weight loss.

Methods of administering one or more Bacillus strain to a piglet arealso provided. Such methods may include feeding the one or more Bacillusstrain to a mother of a piglet. The strain(s) may be fed duringgestation, lactation, or both. The one or more Bacillus strain may alsobe fed to nursery pigs and to grow-finish pigs.

Bacillus Strains:

Bacillus strains have many qualities that make them useful forcompositions that are ingested by animals. For example, Bacillus strainsproduce extracellular enzymes, such as proteases, amylases, andcellulase. In addition, Bacillus strains produce antimicrobial factors,such as gramicidin, subtilin, bacitracin, and polymyxin. Bacillusstrains are also spore formers and thus, are stable. Additionally,several species of Bacillus have GRAS status, i.e., they are generallyrecognized as safe. All B. subtilis strains are GRAS. The Bacillusstrains described herein are aerobic and facultative sporeformers.Bacillus species are the only sporeformers that are considered GRAS.Feeding microorganisms that have GRAS status to livestock is anacceptable practice amongst producers, veterinarians, and others in thelivestock industry.

Bacillus strains that can be used in the methods described hereininclude the following B. subtilis strains: 22CP1, 3AP4, 15AP4, 2084,LSSAO1, and 27. Other Bacillus strains are included within the scopedescribed herein. On Jan. 12, 2005, strains 22CP1, 3AP4, and 15AP4 weredeposited at the American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. 20110-2209 and given accession numbersPTA-6508, PTA-6506 and PTA-6507, respectively. Strains 2084, LSSAO1, and27 were deposited on Mar. 8, 2007, Jan. 22, 2008, and Jan. 24, 2008,respectively, at the Agricultural Research Service Culture Collection(NRRL), 1815 North University Street, Peoria, Ill., 61604 and givenaccession numbers NRRL B-50013, NRRL B-50104, and NRRL B-50105,respectively. All deposits were made under the provisions of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure.

Bacillus strains 22CP1, 3AP4 and 15AP4 were isolated from differentgeographical regions of North America and from different environmentalsources. Specifically, strain 22C-P1 was isolated from a swine lagoonfrom the eastern United States, strain 3AP4 was isolated from chickenlitter from Canada, and strain 15AP4 was isolated from turkey litterfrom the Western United States.

Bacillus strains described herein can be combined, such as in thenon-limiting examples of combinations of Bacillus strains shown in Table1 below and then fed to pigs. A combination can be determined based onthe Clostridium strains present in a specific production facility orother environment. The combination of Bacillus strains can be modifiedif the Clostridium strains change. In addition, because the strains arealso useful in asymptomatic animals, the combination of Bacillus strainsthat are fed can be independent of the Clostridium strains present in aspecific production facility or other environment.

TABLE 1 Formula Strains Counts and Application Rates 25%, 25%, 3AP4,2084, 7.5 × 10⁸ CFU/g of product 25%, 25% 27, LSSAO1 Gestation-1 lb ofproduct per ton of feed Lactation-5.0 lb of product per ton of feed 25%,25%, 3AP4, 15AP4, 1.5 × 10⁸ CFU/g of product 25%, 25% 27, LSSAO1Gestation-1 lb of product per ton of feed Lactation-5.0 lb of productper ton of feed 50%, 50% LSSAO1, 3AP4 7.5 × 10⁸ CFU/g of productGestation-1 lb of product per ton of feed Lactation-5.0 lb of productper ton of feed

Although not intended to be a limitation to the present disclosure, itis believed that inhibition of Clostridium pathogens is accomplished bythe Bacillus strain(s) via the secretion of an active metabolite fromthe Bacillus.

Preparation of the Bacillus Strains:

The Bacillus strains are grown in a liquid nutrient broth, preferably toa level at The counts may be increased or decreased from this number andstill have complete efficacy. CFU or colony forming unit is the viablecell count of a sample resulting from standard microbiological platingmethods. The term is derived from the fact that a single cell whenplated on appropriate medium will grow and become a viable colony in theagar medium. Since multiple cells may give rise to one visible colony,the term colony forming unit is a more useful unit measurement than cellnumber.

The Bacillus strains of the present invention are produced byfermentation of the bacterial strains. Fermentation is started byscaling-up a seed culture. In one embodiment, Bacillus cultures aregrown in growth medium, such as TSB or TSA, for 24 to 48 hrs at 32° C.with agitation in a shaking incubator to a final pH of 7.3±0.2. Thisinvolves repeatedly and aseptically transferring the culture to a largerand larger volume to serve as the inoculum for the fermentation, whichis carried out in large stainless steel fermentors in medium containingproteins, carbohydrates, and minerals necessary for optimal growth. Anon-limiting exemplary medium is TSB. After the inoculum is added to thefermentation vessel, the temperature and agitation are controlled toallow maximum growth. Once the culture reaches a maximum populationdensity, the culture is harvested by separating the cells from thefermentation medium. This is commonly done by centrifugation.

The count of the culture can then be determined and is important whencombined with a carrier. At the time of manufacture, the Bacillus countpreferably is at least about 1.0×10¹¹ CFU/g.

Use of the Bacillus Strains:

The Bacillus strains described herein can be used in direct-fedmicrobials, that is they can be fed directly to swine. In oneembodiment, one or more Bacillus strains is fed to female pig breedingstock during gestation and/or during lactation When one or more Bacillusstrains are fed to female pig breeding stock, the strain(s) istransferred to piglets at least through the oral-fecal route. In anotherembodiment, piglets can be fed one or more Bacillus strains from the dayof birth to weaning at about 17-24 days old. This can be done with anoral drench or any other suitable form of delivering the Bacillusstrains. In other embodiments, nursery pigs are fed one or more Bacillusstrains. The strains can also be fed to grow/finish pigs and to pigs ofdifferent ages.

Administration of one or more Bacillus strains to animals isaccomplished by any convenient method, including adding the Bacillusstrains to the animals' drinking water, to their feed, or to thebedding, or by direct oral insertion, such as by an aerosol. Bacillusstrains preferably are administered as spores.

Bacillus strains described herein may be presented in various forms, forexample as a top dress, liquid drench, gelatin capsule, or gels. In oneembodiment of the top dress form, freeze-dried Bacillus fermentationproduct is added to a carrier, such as whey, maltodextrin, sucrose,dextrose, limestone (calcium carbonate), rice hulls, yeast culture,dried starch, sodium silico aluminate. In one embodiment of the liquiddrench, freeze-dried Bacillus fermentation product is added to acarrier, such as whey, maltodextrin, sucrose, dextrose, dried starch,sodium silico aluminate, and a liquid is added to form the drench. Inone embodiment of the gelatin capsule form, freeze-dried Bacillusfermentation product is added to a carrier, such as whey, maltodextrin,sugar, limestone (calcium carbonate), rice hulls, yeast culture driedstarch, and/or sodium silico aluminate. The Bacillus and carrier areenclosed in a degradable gelatin capsule. In a one embodiment of thegels form, freeze-dried Bacillus fermentation product is added to acarrier, such as vegetable oil, sucrose, silicon dioxide, polysorbate80, propylene glycol, butylated hydroxyanisole, citric acid, andartificial coloring to form the gel. In all of the examples, multiplecarriers can be used.

To prepare direct-fed microbials, the cultures and the carrier can beadded to a ribbon or paddle mixer and mixed preferably for about 15minutes, although the timing can be increased or decreased. Thecomponents are blended such that a uniform mixture of the carrier andcultures result. The final product is preferably a dry, flowable powder.

The following dosages are for all of the Bacillus strains that are fed.That is, if a single strain is fed, then that single strain is used atthe listed dosage. If multiple strains are used, substantially equalamounts of each strain are used for a total dosage that is listed. Forexample, where two strains are used, half of each is used to arrive atthe total dosage.

When fed to female pig breeding stock during gestation, the one or moreBacillus strains with a total microbial count of 7.5×10⁸ CFU/g of DFMincluding one or more strain blended to this count in a carrier is fedat a rate of 1 lb of the DFM per ton of feed, to provide 3.75×10⁵ CFU/gof feed. When fed during lactation, the one or more Bacillus strainswith a total microbial count of 7.5×10⁸ CFU/g of product is fed at arate of 5 lbs of the DFM per ton of feed, to provide 8.5×10⁸ CFU/g offeed. In one embodiment, the DFM is top dressed onto feed. In anotherembodiment, the DFM is blended into the complete feed. The DFM can beadministered in other ways known in the art, at other dosages, and atother stages in the pig's life.

In at least one embodiment of the liquid drench and gel, each has about1×10⁴ to about 1×10¹⁰ CFU/day. In another embodiment of the liquiddrench and gel, each has about 1×10⁸ CFU/day. In at least one embodimentof the top dress, basemix, and premix, each includes about. 7.5×10⁸CPU/g of top dress, basemix, or premix. This can be added to feed at 1lb/ton of feed, resulting in 3.75×10⁵ CFU/g of feed. However, otherdosages can also be used. In one embodiment of a dosage for inclusioninto water, about 1×10³ CFU/animal/day to about 1×10¹⁰ CFU/animal/day isused. Some embodiments of a dosage for inclusion into water use about1×10⁸ CFU/animal/day. While these examples use freeze-dried Bacillus, itis not necessary to freeze-dry the Bacillus before feeding it toanimals. For example, spray-dried, fluidized bed dried, or solid statefermentation Bacillus or Bacillus in other states may be used. Thestrains can also be administered in a wet cell slurry paste, with orwithout preservatives, in concentrated, unconcentrated, or diluted form.

EXAMPLES

The following Examples are provided for illustrative purposes only. TheExamples are included herein solely to aid in a more completeunderstanding of the presently described invention. The Examples do notlimit the scope described herein described or claimed herein in anyfashion.

Example 1

An initial pool of Bacillus strains were obtained from variousenvironmental samples and a library of Bacillus strains. Bacillusstrains were selected that inhibit representative members from theclusters of Clostridium. For this, tubes were seeded, each with arepresentative pathogen from a representative cluster. Supernatant froma Bacillus strain was added to the seeded tubes and incubated. Afterincubation, the optical density (OD) of control and Bacillus supernatanttreated tubes was measured for each pathogen. Colonies of Bacillus thatproduced a lowered OD were then picked, and the Bacillus isolates weregrown.

Clostridium samples were obtained and screened as follows. Clostridiumsamples were obtained from swabs taken from farrowing units. The swabswere taken from piglets, sows, and the environment. The samples wereplated for Clostridium, and colonies were isolated. DNA was isolatedfrom the colonies, and multiplex PCR was performed of the α-toxin geneof Clostridium was used to identify pathogens. To understand thediversity of the Clostridium pathogens, a comparison of the isolates wasperformed using RAPD-PCR. From the RAPD-PCR results, in a dendogram,clusters of Clostridium were identified.

Useful Bacillus strains were selected by identifying representativeClostridium pathogens present in swine production facilities, such asfarrowing units. In general, once Clostridium pathogens were identified,Bacillus strains were screened to determine which of them inhibit growthof the identified pathogens. Bacillus strains that were useful againstboth C. perfringens and C. difficile were identified. Specifically, theBacillus strains were selected and tested as is described below.

A total of 30 presumptive C. perfringens isolates were obtained from thenine rectal swabs. The results of multiplex PCR identified all 30 of theisolates as C. perfringens type A. Twenty-two of the isolates alsocontained the recently identified β2-toxin which has been correlatedwith GI disease in domestic animals. RAPD PCR was performed on thepathogenic isolates to determine the relatedness among the strains. Theresults were then analyzed to construct a dendrogram (shown in FIG. 1),which was used to select isolates for bacteriocin screening. Isolatesconnected to the same branch on the right of the vertical line, drawn at80% similarity, were considered to be members of the same family. Sevenfamilies were observed and are marked with arrows on the left. Thearrows on the right signify which isolates were screened againstBacillus strains described herein.

The Bacillus strains listed in Table 2 highly significantly (>90%)inhibited the growth of all six C. perfringens isolates. These six C.perfringens isolates represent 96.7% of the total diversity observed.Table 2 below includes the results of screening performed on six C.perfringens isolates representative of unique families isolated from theswabs. One family containing only isolate 1-1, which failed to grow forthe assay, was not tested. All six families tested were significantlyinhibited (>50%) by the Bacillus strains listed in Table 2.

TABLE 2 Isolates from swabs % Inhibition C. perfringens Bacillus strainsType A strain 22CP1 15A-P4 3AP4 BS2084 27 LSSA01 1-2 98.2 100 99.1 100100 97.3 1-3 99.2 99.2 97.7 96.2 100 99.2 2-1 100 96.8 98.9 98.9 100 1002-3 91.7 98.1 98.1 98.1 99.1 98.1 6-2 97.7 97.7 94.6 98.5 98.5 97.7 9-395.9 95.1 96.7 95.9 95.9 96.7

Example 2

Clostridium samples were obtained and screened as is described above inExample 1. The Bacillus strains were selected and tested as is describedin this example.

A total of 194 C. perfringens isolates were obtained from six sets ofswab samples. The results of multiplex PCR identified 189 of theisolates as C. perfringens type A, four isolates as C. perfringens typeE, and one as C. perfringens type C. RAPD PCR was performed on thepathogenic isolates to determine the relatedness among the strains. Theresults were then used to construct a dendrogram (shown in FIG. 2),which was used to identify unique families for screening of Bacillusstrains. Isolates connected to the same branch on the right of thevertical line, drawn at 80% similarity, were considered to be members ofthe same family. Each of these families is indicated by an arrow.

Twenty-one representative isolates were chosen for bacteriocin screeningagainst the strains of Bacillus shown in Table 3. The growth of all buttwo C. perfringens isolates was significantly (>50%) inhibited by acombination of the six Bacillus strains. More bacteriocin screening willbe performed to cover all unique families observed to date.

Table 3 below includes the results of bacteriocin screening performed on21 representative isolates of 21 unique families of C. perfringens. Allbut two of the isolates were significantly inhibited (>50%) by the sixBacillus strains shown in Table 3.

TABLE 3 Isolates from swabs % Inhibition Clostridium Identity ofBacillus strains strain C. perfringens 22CP1 15A-P4 3AP4 BS2084 27LSSA01 21-1  Type A 31.1 37.8 60 5.6 0 22.2 28-2  Type A 0 0 0 9.7 0 3.231-1  Type A 17.3 14.7 9.3 6.7 6.7 0 2-2 Type A 98.3 100 33.3 98.3 99.299.2 5-3 Type A 98.5 98.5 97.7 100 99.2 99.2 10-2  Type A 98.3 99.2 37.599.2 100 98.3 3-2 Type A 98.9 98.9 57.9 100 98.9 98.9 4-2 Type A 99.1100 86.4 100 99.1 99.1 6-2 Type A 99.2 99.2 62.5 99.2 99.2 99.2 2-3 TypeA 96.9 97.7 96.2 98.5 97.7 97.7 5-2 Type A 97.3 89.1 96.4 98.2 99.1 98.28-3 Type A 99.1 98.2 98.2 99.1 98.2 97.3 8-4 Type A 96.2 91.5 97.2 99.197.2 98.1 10-1  Type A 98.4 98.4 98.4 96.8 96.8 96 11-2  Type E 97.798.5 98.5 98.5 98.5 98.5 7-1-1 Type E 94.5 96.4 96.4 96.4 95.5 95.57-3-3 Type E 42.3 41.5 50.8 75 42.3 73.8 7-4-1 Type A 97.7 98.5 96.998.5 98.5 97.7 13-1-2 Type A 97.5 96.7 95.8 99.2 98.3 96.7 13-4-2 Type C5.9 17.6 0 31.8 0 77.6 13-9-1 Type A 99.1 99.1 98.2 99.1 99.1 99.1Isolates in the first three rows were not subjected to heat treatment,and are likely contaminated which explains their abnormal resistance tothe Bacillus strains of Table 3. All other swabs from which Clostridiumstrains shown in Table 3 were obtained were heat treated to kill anynon-spore forming contaminants.

Example 3

Clostridium samples were obtained and screened as is described above inExamples 1 and 2. The Bacillus strains were selected and tested as isdescribed in this example.

A total of 40 presumptive C. perfringens isolates were obtained, usingPerfringens Agar selective media, from sixteen rectal swabs. The resultsof multiplex PCR identified all 40 of the isolates as C. perfringenstype A. Thirty-five of the isolates also contained the recentlyidentified β2-toxin which has been correlated with GI disease indomestic animals. RAPD PCR was performed on the pathogenic isolates todetermine the relatedness among the strains. The results were thenanalyzed to construct a dendrogram (shown in FIG. 3), which was used toselect isolates for bacteriocin screening. Isolates connected to thesame branch on the right of the vertical line, drawn at 80% similarity,were considered to be members of the same family. Nine families wereobserved and are marked with arrows. The representative isolatesscreened against the Bacillus are listed in Table 4.

Nine representative isolates were chosen for screening againstbacteriocins produced by the strains of Bacillus listed in Table 4. Thegrowth of all nine isolates was highly significantly (>90%) inhibited,and represent 100% of the diversity observed to date. These resultssuggest that a Bacillus direct fed microbial including these strainswill be effective for the control of C. perfringens in this system.

Table 4 below includes the results of bacteriocin screening performed onnine C. perfringens isolates representative of unique families isolatedfrom the swab samples. All nine families tested were significantlyinhibited (>50%) by the Bacillus shown in Table 4. These nine familiescover all of the diversity observed within this sample set.

TABLE 4 Clostridium isolates from swabs % Inhibition ClostridiumIdentity of Bacillus strains strain C. perfringens 3AP4 27 2-2 Type A97.1 88.9 2-3 Type A 100 100 4-1 Type A 94.5 97.3 4-3 Type A 94.7 96.86-2 Type A 95 97.5 6-3 Type A 98.9 95.8 7-3 Type A 100 100 11-1  Type A97.8 100 11-2  Type A 100 100

Example 4

This example describes piglet performance trials and demonstratesimprovements in scour scores and decreased percent scouring litters withthe administration of Bacillus strains 3AP4 and LSSA01. The strains wereadministered as a dried B. subtilis fermentation product developed.These strains were specifically formulated to be effective againstclostridial disease in neonatal pigs. This study was run to determine ifthese strains may also provide a performance benefit by decreasing theload of pathogenic clostridia in a subclinical herd and to determine theefficacy of these strains for enhancing sow and piglet performanceduring lactation in a commercial sow unit. This research evaluated theperformance enhancing benefits of feeding these strains to sows whosepiglets were known to be infected with Clostridium perfringens type A.

Materials and Methods:

A total of 121 sows (Genetiporc) and their respective litters wereevaluated in a commercial swine production facility. At day 72 ofgestation (last 6 weeks of gestation), sows were allotted to threetreatments by parity group (1 vs. 2+) and 114 day farrowing date. Blocksconsisted of three sows of the same parity group. Three treatments wereadministered to the sows during gestation and lactation: 1) a negativecontrol (n=44) diet devoid of BMD and Bacillus strains 3AP4 and LSSA01,2) a positive control diet as 1) supplemented with BMD (n=39), and 3)as 1) with Bacillus strains 3AP4 and LSSA01 supplementation (n=38).Feed-grade antibiotics (i.e. CTC, Pulmotil, etc.) were not used in thegestation and lactation feeds during the trial period. Experimentaltreatments were top-dressed one time daily to approximate 250 g/ton BMDand 454 g/ton Bacillus strains 3AP4 and LSSA01. The Bacillus strains3AP4 and LSSA01 top-dress was formulated by combining 10 lb of Bacillusstrains 3AP4 and LSSA01 (7.5×10⁸ cfu/g product) and 5 lb of cornstarchto provide the equivalent of 1 lb/ton when 1 teaspoon/day wastop-dressed, which provided 2.25×10⁹ cfu/day. BMD was top-dressedbeginning two weeks prior to lactation until weaning, whereas Bacillusstrains 3AP4 and LSSA01 was top-dressed beginning six weeks prior tolactation until weaning.

Sows were weighed prior to farrowing and at weaning to determine sowbody weight change over the course of the lactation period. Litters werecross-fostered within treatment groups only and to a minimum of 9-10pigs/litter within the first 24 hours post-farrowing. Litter weightswere determined after standardization and at weaning to calculate pigletaverage daily gain (ADG). Scouring litters were recorded to determinethe percentage of litters scouring for each treatment, and scouringseverity was determined for each litter daily based on subjective visualevaluation using a 0 to 3 scale (0=no scours and 3=heavy scours).

Data were analyzed using the PROC MIXED procedure of SAS with litterconsidered the experimental unit with the exception of percentage ofscouring litters and scour severity scores which was analyzed by theKruskal-Wallis nonparametric test.

Results and Discussion:

Treatments impacted (P=0.008) the percentage of scouring litters, suchthat litters born to sows supplemented with Bacillus strains 3AP4 andLSSA01 or BMD had a lower percentage of scouring litters than negativecontrol sows (Table 5). Furthermore, Bacillus strains 3AP4 and LSSA01supplementation tended to result in a lower (P=0.08) percentage ofscouring litters than treatment with BMD. Treatments affected (P=0.01)severity of scours as indicated by litter scour scores, such that sowsprovided Bacillus strains 3AP4 and LSSA01 had the lowest scour severityscore of the three treatments. Furthermore, scour severity tended to beless (P=0.06) in litters born to sows provided Bacillus strains 3AP4 andLSSA01 compared to sows supplemented with BMD.

Conclusions:

These data indicate that BMD and Bacillus strains 3AP4 and LSSA01 areeffective in controlling neonatal piglet scours. In addition, Bacillusstrains 3AP4 and LSSA01 may provide additional benefit over BMD bydecreasing the severity of scouring events.

TABLE 5 Sow and piglet effects of Bacillus strains 3AP4 and LSSA01 orBMD supplementation to gestating sows prior to farrowing. Parity, LitterPiglet scours Treatment n n size, n¹ Severity³ %⁴ Bacillus strains 3AP438 3.66 11.5 0.11 10.5 and LSSA01 (6-wk) BMD (2-wk) 39 4.03 11.1 0.4125.6 Negative control (NC) 44 3.86 10.9 0.57 40.9 Pooled standard errorof 0.36 0.2 — — the mean (SEM) Significance² Treatment 0.779 0.139 0.0110.008 Bacillus strains 3AP4 0.960 0.159 — — and LSSA01 & BMD vs NCBacillus strains 3AP4 0.481 0.156 0.064⁵ 0.088⁵ and LSSA01 vs BMD¹Litter size reflects the average number of pigs/litter followingstandardization. ²Levels of significance (P-values) accorded to the maineffect of treatment and a single degree-of-freedom comparison ofBacillus strains 3AP4 and LSSA01 and BMD supplementation. ³Severity (0 =no scours, 3 = heavy scours) of scouring of pigs in a litter wasanalyzed by the Kruskal-Wallis nonparametric test. ⁴Percentage oflitters exhibiting signs of scouring was analyzed by the Kruskal-Wallisnonparametric test. ⁵ Bacillus strains 3AP4 and LSSA01 and BMD treatmentdata (excluding negative control data) analyzed by the Kruskal-Wallisnonparametric test.

Example 5

This example describes research conducted to document that Bacillussubtilis organisms fed to sows during gestation and lactation weretransferred to suckling piglets. Specifically, this research identifiedthe fecal-oral transfer of Bacillus spores from Bacillus strains 3AP4and LSSA01 into the nursing piglets from supplemented sows. However,other Bacillus sporeformers are believed to also would be transferredvia the fecal-oral route. This research also evaluated the effectivenessof these spores isolated from sow and piglet feces against Clostridiumperfringens.

This example documented the presence of Bacillus spores fromsupplemented sows in their feces and any transfer to their piglets viathe fecal-oral route of transmission. This example also measured theinhibition of Clostridia by Bacillus organisms isolated from sow andpiglet feces from control- and fed sows fed strains 3AP4 and LSSA01.

Materials and Methods:

Two treatments were used: 1) control and 2) strains 3AP4 and LSSA01supplemented to the sow 2 weeks prior to and throughout lactation.Gestation and lactation diets that comprised typical farm managementrations but without feed-grade antibiotics or microbial products wereused as the control basal diets in this experiment. The strains 3AP4 andLSSA01 treatment was top-dressed over the gestation ration administeredto each sow and was mixed, bagged and administered to treated sowsduring the lactation phase. The strains were formulated to provide 3.75CFU/g of feed with each method of application. Nine sows were randomlyassigned to the control treatment and 27 sows represented the treatmentwith strains 3AP4 and LSSA01. Fecal samples (˜100 g) were collected from5 control sows and 15 treated sows before supplementation with strains3AP4 and LSSA01, on d −1 prior to farrowing (d 0), and d 14 afterfarrowing. Samples were also collected from piglets from each sowslitter on d 3, 5, and 14 after farrowing. Fecal samples obtained fromthe pigs within the same litter were pooled into a single sample bag(approximately 25-30 grams of fecal material from the litter). Fecalsamples were obtained fresh and if possible were taken directly from thesow or piglet by rectal stimulation and placed in whirl-pak bags. (Note:piglet feces were removed from the crate on a daily basis. However, sowfeces was not removed from the crate floor.) Fecal samples were platedfor the determination of the presence of strains 3AP4 and LSSA01 throughvisual inspection of colony morphology. Furthermore, Bacillus present inthe fecal samples were isolated and screened against C. perfringensisolates in vitro to determine if the inhibitive activity of theBacillus were maintained after passage through the gastrointestinaltract. Strains 3AP4 and LSSA01 were detected in the fecal samples oftreated sows on d −1 and d 14 and in litters from treated sows on d 5and d 14, documenting the fecal-oral transfer of strains 3AP4 and LSSA01from sow to pig.

Laboratory Analyses:

Fecal Sample Assay-Bacillus Plating.

Eleven grams of the fecal sample was weighed and placed into a whirlpakbag. Samples were masticated with 99 mL of peptone (−1 dilution) andwere spore treated for 10 minutes at 80° C. A −2 dilution was made with1.1 mL of the sample in a 9.9 mL tube of peptone as well as −3 and −4dilutions. The −1 to −4 dilutions were pour-plated in duplicate withTryptic Soy Agar and incubated at 32° C. for 24 hours. Plates from thetreated pigs were counted by hand for strains 3AP4 and LSSA01 strainenumeration.

Fecal Sample Assay—Bacteriocin Screening Assay:

Eleven grams of fecal material was weighed, transferred to a glass tubecontaining appropriate media for Bacillus, and incubated for 24 hours.Following the 24-hour incubation, samples were centrifuged and thesupernatant was collected for use in a bacteriocin screening assay. Thebacteriocin screening was conducted in a 48 well format, testinginhibition of the supernatant produced by Bacillus isolated from fecalmaterial of control and treated sows or pigs against six differentstrains of Clostridium perfringens. The plates were incubated at 37° C.for 24 hours and inhibition of clostridial growth was measured byabsorbance using a plate reader.

Results and Discussion:

Enumeration of Strains 3AP4 and LSSA01 Organisms:

Bacillus strains 3AP4 and LSSA01 were detected in the fecal samples oftreated sows on lactation day −1 and 14, documenting that administrationof strains 3AP4 and LSSA01 Bacillus in the feed inoculates the sows'feces ranging from 10⁵ to 10⁶ cfu/g of feces (Table 6). Strains 3AP4 andLSSA01 organisms could not be detected in the feces of pigs from treatedsows at 3 d of age, however they were present at 5 and 14 d of age atcounts ranging from 10³ to 10⁴ cfu of strains 3AP4 and LSSA01/g ofpiglet feces. Detection of Bacillus strains 3AP4 and LSSA01 in the fecesof sows supplemented with strains 3AP4 and LSSA01 and their pigletsdocuments the transfer of Bacillus strains 3AP4 and LSSA01 from sow topiglet through the fecal-oral route of transmission.

Inhibition of Clostridium perfringens by Strains 3AP4 and LSSA01Organisms:

Total Bacillus isolated from the feces of sows supplemented with strains3AP4 and LSSA01 on d −1 of lactation tended to provide greater (P=0.07)inhibition of C. perfringens growth than Bacillus isolated from thefeces of control sows (Table 7). Likewise, Bacillus isolated from thefeces of 5 d old pigs from Strains 3AP4 and LSSA01 supplemented sowsexhibited greater (P=0.01) inhibition of C. perfringens growth comparedto bacteria isolated from the feces of 5 d old pigs from control sows.

General Conclusions:

These data illustrate the presence of strains 3AP4 and LSSA01 in thefeces of supplemented sows and illustrates the transfer of strains 3AP4and LSSA01 to piglets from litters of supplemented sows. Furthermore,total Bacillus growth from the feces of sows supplemented with strains3AP4 and LSSA01 and their piglets exhibited greater inhibition of C.perfringens growth compared to total Bacillus cultured from the feces ofcontrol sows and their piglets, demonstrating that not only doestransfer of Bacillus strains 3AP4 and LSSA01 occur, but these strains'effectiveness at inhibiting C. perfringens is maintained through thetransfer to the piglet.

TABLE 6 Counts of Strains 3AP4 and LSSA01 (CFU/g and log 10 of feces)from sows and respective litters. Strains 3AP4 and LSSA01 Lactationsupplemented Item Day Control CFU/g log 10 Sow Jan. 29, 2007 −1 N/D 1.1× 10⁶ 5.62 Feb. 19, 2007 14 N/D 3.7 × 10⁵ 5.53 Piglets Jan. 31, 2007 3N/D N/D N/D Feb. 2, 2007 5 N/D 6.0 × 10³ 3.11 Feb. 19, 2007 14 N/D 1.5 ×10⁴ 3.97 N/D = Not detectable

TABLE 7 Percent growth inhibition of six Clostridium perfringensisolates by strains 3AP4 and LSSA01 isolated from fecal samples of sowsand their respective litters. Strains 3AP4 Lactation and LSSA01 Item DayControl supplemented P value^(a) Sow Jan. 29, 2007 −1 38.06 73.56 0.07Piglets Feb. 2, 2007 5 24.37 93.13 0.01 ^(a)Single degree of freedomcontrasts between control and sows supplemented with strains 3AP4 andLSSA01

Example 6

This example shows a decrease in percent scouring litters, improved 15 dpiglet body weight, improved piglet ADG, and decrease in sow weightloss. Bacillus strains 15AP4 and LSSA01 were used in this study todetermine if these strains reduced scours and improved piglet growthperformance pre-weaning in a commercial sow herd subclinical forclostridial disease.

Bacillus strains 15AP4 and LSSA01 were evaluated as a dried Bacillussubtilis fermentation product that was developed to reduce the incidenceof disease associated with pathogenic Clostridium perfringens andClostridium difficile strains. Previous rectal swabs taken at theresearch site where this study was conducted indicate that C.perfringens and C. difficile were present, were susceptible to the 15AP4and LSSA01 Bacillus strain treatments, and may be were contributing tolitter mortality, morbidity, and reduced growth rate pre-weaningalthough the herd was considered subclinical for clostridial disease.This research evaluated if the Bacillus strains 15AP4 and LSSA01 reducedscours and enhanced pre-weaning piglet performance.

Experimental Procedures:

Animals. Data were gathered from 71 sows per treatment of mixed parityand genotype. Sows were randomly allotted to one of two treatments,either control or DFM topdressed with Bacillus strains 15AP4 and LSSA01the last five weeks of gestation and throughout lactation.

Treatment Diets: Gestation and lactation diets were devoid ofantibiotics, and no supplemental nutrition (creep feed, milk replacer,etc.) was provided to piglets during lactation. Topdress treatments withBacillus strains 15AP4 and LSSA01 were administered once each day toprovide each sow with 2.25×10⁹ cfu of the strain combination each day.Sows were topdressed starting on d-79 of gestation (5 weeks prior tofarrowing) and treatment continued throughout lactation.

Litters: Litters were cross-fostered within treatment group within 24hours after farrowing to a minimum of 10 and maximum of 12 pigs/litter.Litters were individually weighed at standardization and 15 dayspost-standardization.

Sow Performance and Subsequent Litter Data: Identification number,genotype, parity, replicate number, farrowing date, treatment startdate, wean date, date of standardization, and date of litter weight wererecorded for each sow on test. Native litter information for each sowwas also recorded, including number born alive, stillbirths, andmummies. Sow body condition was determined at standardization and 15days post-standardization. Body condition was recorded based on asubjective 3-point scale, with 1=thin, 2=desired condition, and3=overweight.

Scour Scores: The percentage of scouring litters was calculated as thetotal number of litters identified as scouring/total number of littersper treatment×100. Litters were only considered scouring during thefirst two days after farrowing if the scours persisted to d 3post-farrowing to eliminate any consideration of loose stools due to“milk scours” that clear up within the first two days after birth. Eachlitter was assigned a subjective scour score daily during lactationdefined as follows: 0=no scours present in the litter; 1=less than 50%of the litter exhibited signs of scours; 2=More than 50% of the litterexhibited signs of scours.

Experimental Design: Data were analyzed as a randomized complete blockdesign with two treatments and sow (or litter) as the experimental unitusing the PROC MIXED procedure of SAS.

Results and Discussion:

Control sows tended (P=0.08) to have more live pigs born than sowssupplemented with Bacillus strains 15AP4 and LSSA01 (Table 8). Sowsallotted to the treatment with Bacillus strains 15AP4 and LSSA01 enteredthe lactation phase with a slightly lower body condition score thancontrol sows but had a higher body condition score 15 d after farrowing,indicating sows supplemented with Bacillus strains 15AP4 and LSSA01 lostless weight during the lactation period as evidenced by thesignificantly lower (P=0.006) change in body condition score valuecompared to control sows.

TABLE 8 The effect of the DFM supplementation pre-farrowing on littersize and body condition score of sows. Body Condition Score¹ Live Still-Ch. Treatment n Parity, n born, n born, n Mum., n d 0 D 15 BCS DFM 714.97 10.82 1.65 0.35 1.99 1.96 0.028 Negative control 71 4.80 11.78 1.480.34 2.08 1.86 0.225 (NC) Pooled standard 0.17 0.57 0.68 0.11 0.05 0.060.058 error of the mean (SEM) Significance (P 0.47 0.08 0.44 0.91 0.130.23 0.006 value) ¹Subjective body condition score based on a 3-pointscale: 1 = thin; 2 = good; 3 = overweight.

Supplementation with Bacillus strains 15AP4 and LSSA01 to sows resultedin a tendency toward a greater (P=0.07) average piglet body weight 15 dafter farrowing compared to control sows (Table 9). The improvement in d15 body weights of pigs born to sows supplemented with Bacillus strains15AP4 and LSSA01 was further supported by a tendency toward greater(P=0.11) ADG compared to control pigs. The coefficient of variationassociated with individual piglet body weight within a litter tended tobe lower (P=0.06) in litters born to sows supplemented with Bacillusstrains 15AP4 and LSSA01 compared to control sows at birth (d 0), withthis trend remaining evident numerically (P=0.27) 15 d after farrowing.

TABLE 9 The effect of DFM on piglet performance and litter variation.d-0 Pig d-0 Pig d-15 Pig ADG, Mortality + Treatment n wt., lbs CV, %wt., lbs d-15 CV, % lbs/d Morbidity DFM 71 3.62 13.34 11.99 16.38 0.549.62 Negative control 71 3.55 14.45 11.50 17.27 0.51 9.75 (NC) Pooledstandard 0.077 0.409 0.24 0.60 0.015 1.43 error of the mean (SEM)Significance (P 0.18 0.06 0.07 0.27 0.11 0.94 value)

Supplementation with Bacillus strains 15AP4 and LSSA01 decreased(P=0.02) the percentage of scouring litters from 14% to 2.8%, andlitters born to sows provided the DFM had lower (P=0.02) average scourscores in the first week after birth compared to control litters (Table10).

TABLE 10 The incidence of drug treatment and scour in litters reared bysows supplemented DFM 5-weeks prior to farrowing and during a 15-dlactation period. Ave. d0-7 Litters Litters Trts. given scour scouring,treated for in scouring Treatment n score¹ %² scours, %³ litters, n⁴ DFM71 0.012 2.81 2.82 11.0 Negative control 71 0.069 14.08 7.04 16.4 (NC)Significance 0.02 0.02 0.25 0.54 (P value) ¹Subjective scour score: 0 =no scours; 1 = less than 50% of litter showing scours; 2 = More than 50%of the litter showing signs of scour. ²Calculated as the total number oflitters identified as scouring/total number of litters per treatment.³Calculated as the number of litters treated for scours (Tylan)/totalnumber of litters per treatment ⁴Average number of Tylan injectionsgiven per litter.

Example 7

Introduction:

A Bacillus-based direct-fed microbial (DFM) was specifically developedto aid in the prevention of clostridial-related scours in neonatal pigs.In vitro analysis of the effects of the DFM strongly supports itseffectiveness against C. perfringens and C. difficile isolated fromscouring piglets. This study was devised to document sow and litterperformance responses and decreases in piglet intestinal clostridiacounts from the supplementation of the DFM to sows in an asymptomaticherd.

Experimental Procedures:

A total of 208 mixed parity sows (and some gilts) were used for theexperiment and were fed one of two dietary treatments during thegestation and lactation periods. One hundred and four sows were fedstandard control diets during gestation and lactation, and theadditional 104 sows were fed the standard diets supplemented with a DFMincluding substantially equal CFU counts of Bacillus strains LSSA01 and3AP4 at a 1 lb/ton inclusion level of product containing 7.5×10⁸ CFU/gof both of the strains for six weeks prior to and throughout thelactation period. Treatments were distributed in a randomized completeblock design with sows blocked by parity with the average parity being1.78 for DFM sows and 1.82 for control sows. Pulmotil (and otherantibiotics) were excluded from the experimental diets. Lactation lengthwas targeted at 21 days, but lengths from 18 to 25 days were deemedacceptable.

Daily feed intakes are an estimate based on feed drop information. In aprevious experiment actual feed drop weight was regressed againstcalibrated feed drop weight and gave the equation y=0.6181×^(1.2357)R²=0.9882, where y=actual output and X=calibrated drop weight Datacollected on their litters included, 1) number born and number bornalive, 2) number of stillborns and mummies, 3) number weaned, 4) averagepiglet weight at birth and weaning, 5) ADG during the lactation period,6) scour scores, which are presented as a percentage of litters scouringduring the first week of age. Scores range from 0 to 5 with 0 beingnormal and 5 being severe. There were no scores above 2 reported forthis study, and 7) pre-weaning mortality.

From the individual piglets, data collected included 1) individualpiglet body weights on the last two farrowing groups—litter variation,2) enumeration of DFM Bacillus in the GI tract of piglets at 3 d of age,and 3) enumeration of clostridia in the GI tract of piglets at 3 and 10d of age. A total of 50 litters (523 piglets) for DFM sows and 48litters (523 piglets) for control sows were used for data analysis.

One piglet per litter was selected from the litters selected pertreatment for sampling on d 3 of age and d 10 of age from each farrowinggroup. On each sampling day, piglets were euthanized by electricalstunning and exsanguinated for collection of gastrointestinal tissuesfor bacterial enumeration. Sampling occurred in as many of the fourfarrowing groups as needed to obtain adequate replication [˜15pigs/treatment for each age (d 3 and d 10)].

Sampling and Dissection: Following euthanization by exsanguination,piglet intestinal samples were dissected to obtain gastrointestinaltissue samples. Specific sections were obtained as follows: The duodenalsection for enumeration of clostridia and Bacillus counts were ligatedat the pyloric junction and 10 cm distally, dissected and placed in aWhirl-Pak bag with ˜10 mL of sterile saline. The jejunal sample forclostridia and Bacillus enumeration was ligated 40 cm distally from theduodenum end and 20 cm distally from the first ligation, dissected andplaced in a Whirl-Pak bag with ˜10 mL of sterile saline. The ilealsection for clostridia and Bacillus enumeration was ligated at theileal-cecal junction and 15 cm proximally, dissected, and placed in aWhirl-Pak bag with ˜10 mL of sterile saline. A section of the distalcolon was obtained by ligating the colon section at its connection tothe spiral colon and 8 cm distally for clostridia and Bacillusenumeration. The ligated section was dissected and placed in a Whirl-Pakbag with ˜10 mL of sterile saline. Samples were transported to theAgtech lab on ice for processing.

Enumeration of intestinal Clostridia and Bacillus. Gastrointestinalsamples were plated to enumerate DFM Bacillus strains in 3 d old pigletsas well as clostridia counts in both 3 and 10 d old piglets in the smalland large intestine. Samples were dissected on-farm as described in theprevious section. Two dilutions of each intestinal section (duodenum,jejunum, ileum, and large intestine) were plated in duplicate on TrypticSoy Agar (TSA) for Bacillus growth and C. perfringens Agar (CPA) forclostridia growth. Each sample was rinsed with sterile peptone to removeintestinal contents, dissected longitudinally, weighed, diluted in 99 mLof peptone and masticated for 60s. After mastication, a 10 ml aliquotwas taken from the −1 dilution of each tissue sample and placed in asterile test tube. Samples were spore treated in a heat block at 70° C.for 30 minutes, after which a subsequent −3 dilution was made. The −1and −3 dilutions were plated onto TSA for the enumeration of Bacilluscolonies and onto CPA to determine clostridial counts. The cells fromthe −1 dilution were pelleted by centrifugation, resuspended in 10 ml ofTSB+10% glycerol, divided into two aliquots in 15 mL conical tubes, andfrozen for subsequent analysis.

Results and Discussion:

Daily feed intake did not differ when comparing control and DFMsupplemented sows (Table 11). DFM supplemented sows farrowed more(P>0.05) total pigs and pigs born alive than control sows, although thisresponse is likely a reflection of conception rate that occurred beforeDFM treatment. Sows supplemented with DFM weaned more pigs (P=0.06) thancontrol sows and, although not significant (P=0.12), this response wasreflected in the lower percentage of piglet mortality with DFMsupplementation.

TABLE 11 Performance of Sows Supplemented with DFM in Late Gestation andLactation^(1,2,3) Wean-to- Daily feed Estrus Treatment intake, lbs⁴Interval, d⁵ DFM 12.22 5.90 Control 11.99 5.60 SEM 0.37 0.60 Level of0.54 0.55 significance Dietary treatment ¹The average parity was 1.78for DFM sows and 1.82 for control sows. ²Feed intake: n = 99 for DFMsows and n = 101 for control sows; Wean-to-estrus interval: n = 91 forDFM sows and n = 95 for control sows. ³Outliers defined as any datapoint plus or minus three standard deviations from the mean. ⁴Averagefeed intake over lactation period. Daily feed intake is an estimatebased on feed drop information (calculator determined in JBS Unitedexperiment 07-S025). ⁵There was not effect f lactation length onwean-to-estrus interval.

Piglet performance determined by weighing pigs individually from asubset of 50 litters from DFM supplemented sows and 48 litters fromcontrol sows is displayed in Table 12. No differences in pigletperformance (weaning weights, ADG, litter size, and mortality) wereobserved between treatments when analyzing the data from this subset oflitters. However, when piglet performance was evaluated based on litterand encompassing the entire dataset from the trial (103 DFM litters and102 control litters) litters nursing DFM supplemented sows had greater(P=0.02) weaning weights compared to control litters (Table 13).Although initial litter weight was also greater (P=0.01) for littersnursing DFM supplemented sows, the improvement in litter weaning weightwas at least partially due to the tendency toward improvement (P=0.09)in litter ADG of DFM litters compared to controls.

TABLE 12 Native Litter Performance of Pigs Nursing Sows Supplementedwith DFM in Late Gestation and Lactation.^(1,2,3) Treatment Total bornBorn alive Stillborn Mummies DFM 13.27 12.15 1.03 0.28 Control 12.3911.09 1.21 0.14 SEM 0.43 0.37 0.17 0.09 Level of significance 0.04 <0.010.27 0.11 Dietary treatment ¹The average parity was 1.78 for DFM sowsand 1.82 for control sows. ²Total born: n = 104 for DFM and controlsows; Born alive and mummies: n = 103 for DFM and control sows;Stillborn: n = 101 for DFM and control sows; ³Outliers defined as anydata point plus or minus three standard deviations from the mean.

The high percentage of scouring litters was surprising as the healthstatus of the herd at the time of the trial was considered to berelatively healthy (Table 13). More surprising was the numericallyhigher (P=0.64) percentage of DFM litters scouring compared to controls,as data from previous trials have consistently reported decreases in thepercentage of scouring litters with administration of DFM. Scourseverity was also scored on a scale of 0 (no scours) to 5 (severescours), and none of the litters on test scored above a scour severityscore of 2, suggesting these scours were likely transient “milk” scoursand not indicative of a major health challenge.

TABLE 13 Standardized Litter Performance (Expressed Per Piglet and PerLitter) of Sows Supplemented with DFM in Late Gestation andLactation^(1,2,3) Per Piglet Per Litter Initial Weaning Weaning weight,weight, Gain, ADG, Standardized Initial weight, Gain, ADG, Wean lbs lbslbs lbs Litter Size weight, lbs lbs lbs lbs % Scours⁴ litter size %mortality DFM 3.39 13.82 10.44 0.53 10.99 37.14 136.27 99.33 5.01 31.449.86 10.41 Control 3.09 13.43 10.41 0.52 10.95 33.97 128.76 94.91 4.7428.66 9.56 12.76 SEM 0.12 0.25 0.25 0.01 0.06 1.28 3.15 3.21 0.16 5.860.16 1.54 Level of 0.01 0.11 0.88 0.56 0.54 0.01 0.02 0.17 0.09 0.640.06 0.12 significance Dietary treatment ¹Outliers defined as any datapoint plus or minus three standard deviations from the mean. ²Lactationlength ranged from 18 to 25 days. ³Growth performance per piglet n = 101for DFM litters and n = 100 for control litters; Growth performance perlitter: n = 103 for DFM pigs and n = 102 for control pigs; Scour scores:n = 104 for DFM and control pigs; Standardized litter size: n = 99 forDFM pigs and n = 98 for control pigs; Mortality: n = 99 for DFM andcontrol pigs. ⁴Percent of litters scouring at least once during wk 1 ofage; n = 52 litters for DFM piglets, n = 48 litters for control piglets.Scours were scored on a scale from 0 to 5 with 0 being no scours and 5being severe scours. There were no observations above a 2 reported forthis study.

Bacillus organisms from the DFM were not detected in the intestinaltract of piglets sampled at 3 days of age. Intestinal clostridia countsfrom a combination of ileal and distal colon samples obtained from 3 dayold piglets were reduced in piglets nursing DFM supplemented sowscompared to control sows (2.9×10⁵ vs 1.0×10⁶±4.6×10⁵, P=0.07). AlthoughDFM supplementation did not impact clostridia counts in the ileum ofpiglets at 3 days of age, clostridia counts were reduced (P>0.05) in thedistal colon of pigs nursing DFM supplemented sows compared to controlpigs (section×treatment interaction, P=0.14; FIG. 4). Althoughclostridia counts in the intestinal tract of 10 d old pigs nursing DFMsupplemented sows were numerically lower compared to control pigs, therewas no statistical difference between treatments (1.5×10⁵ vs.3.3×10⁵±1.6×10⁵, P=0.27).

In summary, this study demonstrates the benefits of DFM supplementationto sows during gestation and lactation on number of pigs weaned andpiglet weight gain in a herd considered to have a subclinical clostridiachallenge. Additionally, the DFM decreased the clostridial load in thegastrointestinal tract of 3 day old piglets by specifically decreasingthe clostridial load in the large intestine. These data support previousfindings in which DFM improved sow and piglet performance in herdswithout clinical clostridia challenges.

Example 8

Objective:

To determine the efficacy of a direct-fed microbial (DFM) for enhancingsow and piglet performance during lactation in a commercial sow unitconsidered subclinical for clostridia scours.

Materials and Methods:

Sows (Genetiporc) and their respective litters were evaluated in a1200-sow commercial swine production facility. At day 72 of gestation(last 6 weeks of gestation), sows were allotted to three treatments byparity group (1 vs. 2+) and 114 day farrowing date. Blocks consisted ofthree sows of the same parity group. Three treatments were administeredto the sows during gestation and lactation: 1) a negative control dietdevoid of BMD and a DFM (described below), 2) a positive control dietconsisting of the negative control supplemented with BMD, and 3) thenegative control diet with DFM supplementation. Feed-grade antibiotics(e.g., CTC, Pulmotil) were not used in the gestation and lactation feedsduring the trial period. Also, litters were not treated with oral orinjectable antibiotics for the duration of the trial unless there weresevere health challenges. If treatments occurred, all individual pigtreatments were recorded. Sows were vaccinated two weeks prior tofarrowing and treatments were top-dressed one time daily to approximate250 g/ton BMD and 454 g/ton DFM (Table 14). The DFM includedsubstantially equal CFU counts of Bacillus strains LSSA01 and 3AP4. TheDFM top-dress was formulated by combining 10 lb of DFM (7.5×10⁸ cfu/gproduct) and 5 lb of cornstarch to provide the equivalent of 1 lb/tonwhen 1 teaspoon/day was top-dressed, which provided 2.25×10⁹ cfu/day.BMD was top-dressed beginning two weeks prior to lactation untilweaning, whereas the DFM was top-dressed beginning six weeks prior tolactation until weaning. To avoid cross-contamination in gestation, sowswere grouped together by treatment. A “non-test” sow was placed betweentreatment groups. Any residual feed from sows that were “off-feed” wasremoved from the trough prior to watering to prevent contamination.

TABLE 14 Summary of treatments administered, vaccinations, and top-dressprocedures performed during the experiment. Treatments Vaccination¹ BMD²DFM³ Negative Control Yes No No Positive Control Yes 1 scoop/day BMD, No(BMD) last 2 weeks of gestation through weaning DFM Top-Dress Yes No 1teaspoon/day, last 6 weeks of gestation through weaning ¹Vaccinationincluded Beacon Scour Block (E-coli-4 + Clostridium perfringens type C)distributed by Newport Laboratories. Gilts were vaccinated 4 and 2 weeksprior to farrowing. Sows were vaccinated 2 weeks prior to farrowingonly. ²Each scoop was equivalent to 250 g/ton of BMD. ³Each teaspooncontained 3 g of DFM and provided 2.25 × 10⁹ cfu/day. This wasequivalent to 454 g of the DFM/ton of feed.

Sows were weighed prior to farrowing and at weaning to determine sowbody weight change over the course of the lactation period. Litters werecross-fostered within treatment groups only and to a minimum of 9-10pigs/litter within the first 24 hours post-farrowing. Scouring litterswere recorded to determine the percentage of litters scouring for eachtreatment, and scouring severity was determined for each litter dailybased on visual evaluation using a 0 to 3 scale (0=no scours and 3=heavyscours). The following measurements were recorded for each litter:

Measurements: The following measurements were taken: 1. date farrowed,2. parity, 3. sow body weight (pre-farrow and weaning), 4. no. pigs atstandardization and at weaning, 5. pig removals and reason for removal,7. individual pig treatments, and 8. scouring litters.

Results and Discussion:

Average parity of sows during the second trial was between 4 and 5 forall three treatment groups (Table 15). Although there was no significanttreatment effect (P=0.60), DFM supplemented sows averaged numericallyfewer pigs born alive than control sows or those supplemented with BMD.DFM and BMD supplemented sows had lighter (P=0.02) prefarrowing bodyweights than negative control sows, whereas there was no difference inbody weight between the treatments when sows were weighed 16 d into thelactation period. As a result, DFM and BMD supplemented to sows reduced(P=0.08) body weight loss during lactation compared to control sows. Sowweight loss during lactation was also evaluated with prefarrow bodyweight and live born pigs as covariates in the analysis, and while thisaltered degrees of significance it did not change interpretation of thedata. Percentage of scouring litters and scour severity are notreported, as only one litter was observed to be scouring during the timethis trial was conducted.

TABLE 15 Effects of DFM or BMD supplementation to gestating sows priorto farrowing. d-16 Prefarrow lactation Sow Sow Sow Live weight, sowweight, Weight weight weight Treatment N Parity Born lbs lbs loss, lbsloss, lbs loss, lbs DFM (6-wk) 49 4.88 10.80 544.41 509.04 35.27 36.0636.57 BMD (2-wk) 49 4.40 11.45 543.52 496.38 45.12 46.04 44.60 Negativecontrol 52 4.79 11.20 569.23 521.36 50.35 48.53 50.25 (NC) Covariate —Prefarrow Live sow Born weight Pooled standard 0.41 0.56 13.30 12.146.96 6.70 6.62 error of the mean (SEM) Significance¹ 0.65 0.60 0.08 0.190.08 0.16 0.12 Treatment DFM vs BMD 0.39 0.32 0.95 0.37 0.18 0.17 0.26DFM & BMD vs 0.73 0.88 0.02 0.11 0.08 0.21 0.09 NC ¹Levels ofsignificance (P-values) accorded to the main effect of treatment and asingle degree-of-freedom comparison of DFM and BMD supplementation.

The lack of scouring litters in this second trial documents the varyinghealth status of the herd at the time the two trials were conducted. Theherd had just gone through a PRRS break prior to the start of the firsttrial, whereas the herd was PRRS stable at the time the second trial wasconducted.

Conclusion:

These data indicate that BMD and DFM decreased sow weight loss duringlactation when herd health status was good and when clinical signs ofclostridial scours were not present. This trial and the results of theprevious trial described in Example 4 conducted during health challenges(PRRS and scours) indicate a beneficial effect of DFM supplementationregardless of herd health status.

Example 9

Objective:

To determine the optimal level of supplementation with a direct-fedmicrobial (DFM) in nursery pigs that results in enhanced growthperformance.

Materials and Methods:

There were five (5) dietary treatments with twelve (12) replicates pertreatment and eight pigs per pen. Pigs were allotted by weight and sex.Sex ratio was equal within each replicate. A basal diet was prepared foreach nursery phase. The direct-fed microbial (DFM) included strains 3AP4and LSSA01 in substantially equal amounts of CFUs. The DFM contained3.75×10⁸ CPU/g of each strain for a total of 7.5×10⁸ CFU/g of DFMproduct. Additional ingredients in the DFM product that served ascarrier included calcium carbonate and rice hulls. Diets typical ofthose used in the commercial swine industry were fed during each nurseryphase. Five premixes labeled A through E were prepared for eachtreatment and were used throughout the experiment to prepare eachtreatment diet. Treatment diets defined below were formulated byblending the appropriate premix into the basal diet at the expense ofcorn.

The treatments were as follows: control, control diet+0.50 lb ofproduct/ton of feed, control diet+1.00 lb of product/ton of feed,control diet+2.00 lb of product/ton of feed, control diet+4.00 lb ofproduct/ton of feed. The duration of the treatments was 6 weeks, andthere were four phases: Phase 1 (6 to 8 d), Phase 2 (1 wk), Phase 3 (1wk), and Phase 4 (3 wk).

The following measurements were taken: feed samples at time ofmanufacture, individual pig weights for allotment and weekly pen weightsthereafter, feed intakes weekly, mortality and morbidity, medicinaltreatments, scour scores, and feed samples collected out of feeders.Scour scores were recorded using the following scale: 1=no scours, 2=anylooseness evident in pen, and 3=considerable looseness in pen.

EXCENEL® antibiotic (Pharmacia & Upjohn Co., North Peacock, N.J.) wasadministered on the day of weaning. Mecadox® antibiotic (Phibro AnimalHealth, Ridgefield Park, N.J.) was supplemented in the diet during thenursery phases.

Results and Discussion:

Supplementation of DFM to nursery diets resulted in a quadratic (P<0.03)average daily gain (ADG) response during weeks 3 and 5 of the nurseryperiod (Table 14). This improvement in ADG translated to a quadraticincrease (P<0.05) in pig body weight at the end of weeks 3, 5, and 6,and pig body weight tended (P<=0.08) to increase linearly during week 4with DFM supplementation. Average daily feed intake increasedquadratically (P<0.02) during week 3, with the greatest feed intakeresponse observed when DFM was supplemented at 0.5 lb/ton. Although thegreatest feed intake during week 5 was observed when pigs were fed dietssupplemented with 0.5 lb/ton of DFM, the only treatment that differedfrom the control was a decrease (P<0.05) in feed intake when pigs weresupplemented with DFM at the 4 lb/ton inclusion level.

Contrasts comparing the various levels of DFM supplementation that weretested in this study indicated that DFM supplementation at 0.5 and 1lb/ton of feed resulted in greater (P≦0.05) ADG, average daily feedintake (ADFI) and pig body weight in week 3 when compared to the averageresponses when DFM was supplemented at higher levels (Table 14). Duringweeks 4 and 5, supplementation with DFM at 0.5 lb/ton resulted ingreater (P≦0.05) ADFI and pig body weight compared to the average ofpigs fed DFM supplemented at higher levels, whereas the same was truefor ADG and pig body weight when DFM was supplemented at 1 lb/ton inweek 5 (P≦0.05). Comparisons of DFM supplementation of 2 lb/ton verses 4lb/ton tended (P≦0.10) to indicate general decreases in growthperformance within weeks 2, 3, 5, and 6 when DFM was supplemented at 4lb/ton.

Cumulatively over the entire nursery trial, ADG and pig body weightincreased quadratically (P<0.05) with increasing DFM supplementation,such that supplementation with 0.5 lb/ton yielded the greatest response(Table 15). Average daily feed intake increased linearly (P=0.05) as DFMsupplementation increased with the greatest ADFI occurring in pigssupplemented with DFM at 0.5 lb/ton. Contrasts comparing levels of DFMsupplementation in the overall trial indicated ADG and ADFI were greater(P<0.05) when pigs were fed DFM at 0.5 lb/ton compared to DFMsupplementation at higher levels. Scour scores were generally low inthis study, indicating minimal enteric challenge, and were not impactedby DFM supplementation (Table 15).

In conclusion, DFM supplementation during the nursery period improvedgrowth performance in the middle and later nursery phases, as well asover the cumulative trial. These improvements were evidenced when DFMwas supplemented at the 0.5 and 1.0 lb/ton inclusion level.Specifically, DFM supplemented at 0.5 or 1.0 lb/ton improved ADG as aresult of increased ADFI, and culminated in an approximately 1 lbheavier pig at the end of the nursery period.

TABLE 14 Efficacy of graded levels of DFM on weekly growth performancein nursery pigs¹ Pooled standard Treatment error of Contrast 0.5 lbs/1.0 lbs/ 1 2 3 4 5 the P-values ton ton 2.0 lbs/ton DFM (lbs/ton) meanDiet P- vs. vs. vs. 0 0.5 1.0 2.0 4.0 (SEM) value Linear Quadratichigher higher 4.0 lbs/ton Week 1 Start wt. (lbs) 15.37 15.38 15.44 15.4215.30 0.515 0.412 0.516 0.096 1.00 0.236 0.118 End wt. (lbs) 16.05 16.0315.94 16.07 15.69 0.584 0.649 0.280 0.502 0.562 0.797 0.186 ADG (lb)0.10 0.09 0.07 0.09 0.06 0.026 0.813 0.375 0.806 0.596 0.949 0.338 ADFI(lb) 0.19 0.18 0.17 0.19 0.16 0.016 0.553 0.431 0.902 0.988 0.604 0.187F/G (lb/lb) 1.90 2.00 2.43 2.11 2.67 0.870 0.325 0.359 0.665 0.295 0.2090.363 Week 2 End wt. (lbs) 21.07 21.16 20.91 21.27 20.51 0.753 0.4430.298 0.340 0.454 0.955 0.084 ADG (lb) 0.72 0.74 0.73 0.74 0.69 0.0360.541 0.384 0.204 0.432 0.650 0.137 ADFI (lb) 0.74 0.75 0.74 0.76 0.700.035 0.362 0.240 0.210 0.443 0.687 0.064 F/G (lb/lb) 1.03 1.03 1.021.03 1.03 0.027 0.995 0.967 0.712 1.00 0.717 0.981 Week 3 End wt. (lbs)27.53^(ab) 28.18^(a) 27.88^(a) 27.43^(ab) 26.61^(b) 0.909 0.037 0.0250.023 0.039 0.051 0.109 ADG (lb) 0.902^(ab) 0.968^(a) 0.949^(a)0.858^(b) 0.858^(b) 0.054 0.004 0.014 0.028 0.007 0.004 1.000 ADFI (lb)1.15^(ac) 1.23^(b) 1.18^(ab) 1.12^(ac) 1.08^(c) 0.060 0.003 0.004 0.0150.001 0.016 0.302 F/G (lb/lb) 1.29 1.27 1.24 1.32 1.27 0.031 0.418 0.8990.751 0.804 0.150 0.196 Week 4 End wt. (lbs) 36.00^(ab) 37.03^(b)36.27^(ab) 35.64^(ab) 35.00^(b) 1.138 0.216 0.081 0.160 0.059 0.2020.450 ADG (lb) 1.03 1.11 1.05 1.01 1.05 0.049 0.412 0.679 0.630 0.1030.680 0.438 ADFI (lb) 1.50 1.60 1.53 1.46 1.51 0.070 0.192 0.336 0.5240.044 0.331 0.347 F/G (lb/lb) 1.47 1.45 1.46 1.45 1.44 0.026 0.932 0.5060.993 0.885 0.623 0.777 Week 5 End wt. (lbs) 45.07^(ab) 46.28^(a)45.80^(a) 44.73^(ab) 43.19^(b) 1.334 0.039 0.022 0.026 0.049 0.038 0.128ADG (lb) 1.30^(a) 1.33^(a) 1.33^(a) 1.28^(a) 1.18^(b) 0.041 0.025 0.0140.020 0.132 0.019 0.053 ADFI (lb) 1.91^(ab) 1.97^(a) 1.90^(ab) 1.84^(bc)1.78^(c) 0.054 0.034 0.006 0.166 0.012 0.091 0.324 F/G (lb/lb) 1.49 1.491.44 1.45 1.52 0.026 0.197 0.795 0.047 0.506 0.160 0.065 Week 6 End wt.(lbs) 55.56^(ab) 56.69^(a) 56.40^(a) 55.66^(ab) 53.57^(b) 1.60 0.1340.082 0.044 0.170 0.106 0.102 ADG (lb) 1.50 1.51 1.51 1.53 1.44 0.0550.670 0.517 0.267 0.809 0.616 0.159 ADFI (lb) 2.24 2.31 2.29 2.24 2.180.082 0.651 0.342 0.242 0.350 0.285 0.536 F/G (lb/lb) 1.49 1.53 1.511.47 1.52 0.020 0.281 0.812 0.964 0.213 0.424 0.109 ¹There were 12replicates pens per dietary treatment. ^(a-c)Means with differentsuperscripts within a row are significantly different (P < 0.05)

TABLE 15 Efficacy of graded levels of DFM on cumulative growthperformance and scour scores in nursery pigs^(1,2,3) Pooled standardTreatment error of Contrast 0.5 lbs/ 1.0 lbs/ 1 2 3 4 5 the P-values tonton 2.0 lbs/ton DFM (lbs/ton) mean Diet P- vs. vs. vs. 0 0.5 1.0 2.0 4.0(SEM) value Linear Quadratic higher higher 4.0 lbs/ton Cumulative Startwt. (lbs) 15.37 15.38 15.44 15.42 15.30 0.515 0.412 0.516 0.096 1.000.236 0.118 End wt. (lbs) 55.56^(ab) 56.69^(a) 56.40^(a) 55.66^(ab)53.57^(b) 1.60 0.134 0.082 0.044 0.170 0.106 0.102 ADG (lb) 0.91^(ab)0.95^(a) 0.93^(a) 0.91^(ab) 0.88^(b) 0.033 0.094 0.065 0.043 0.047 0.1320.171 ADFI (lb) 1.27^(ab) 1.33^(a) 1.29^(ab) 1.26^(ab) 1.23^(b) 0.0460.097 0.050 0.113 0.019 0.186 0.442 F/G (lb/lb) 1.40 1.40 1.38 1.38 1.410.009 0.174 0.790 0.066 0.283 0.382 0.050 Scour Scores Week 1 score 1.361.39 1.51 1.62 1.40 0.104 0.352 0.328 0.193 0.290 1.000 0.137 Overallscore 1.14 1.11 1.18 1.14 1.13 0.030 0.530 0.921 0.665 0.192 0.244 0.856¹There were 12 replicates pens per dietary treatment. ²Scour scores canwere recorded as 1 = no scours; 2 = moderate looseness; 3 = considerablelooseness. ³Pigs were fed dietary treatments from weaning to 42 dpost-wean. ^(a-b)Means with different superscripts within a row aresignificantly different (P < 0.05).

It is understood that the various preferred embodiments are shown anddescribed above to illustrate different possible features describedherein and the varying ways in which these features may be combined.Apart from combining the different features of the above embodiments invarying ways, other modifications are also considered to be within thescope described herein. The invention is not intended to be limited tothe preferred embodiments described above, but rather is intended to belimited only by the claims set out below. Thus, the inventionencompasses all alternate embodiments that fall literally orequivalently within the scope of these claims.

1. A method comprising administering to a pig an effective amount toinhibit Clostridium in litters borne to the pig of at least two Bacillusstrains selected from strains 22CP1 (ATCC PTA-6508), 3AP4 (ATCCPTA-6506), 15AP4 (ATCC PTA-6507), 2084 (NRRL B-50013), LSSAO1 (NRRLB-50104), and 27 (NRRL B-50105), wherein at least one strain is LSSAO1(NRRL B-50104).
 2. The method of claim 1, wherein the Bacillus strainsare strains 3AP4 (ATCC PTA-6506) and LSSAO1 (NRRL B-50104).
 3. Themethod of claim 1, wherein the Bacillus strains are strains 15AP4 (ATCCPTA-6507) and LSSA01 (NRRL B-50104).
 4. The method of claim 1, whereinthe Bacillus strain is administered to female breeding stock.
 5. Themethod of claim 1, wherein the Bacillus strain is administered duringgestation of the pig.
 6. The method of claim 5, wherein a totalmicrobial count of 3.75×10⁵ CFU/g of feed/day is administered.
 7. Themethod of claim 1, wherein the Bacillus strain is administered duringlactation of the pig.
 8. The method of claim 7, wherein a totalmicrobial count of 8.5×10⁸ CFU/g of feed/day is administered.
 9. Themethod of claim 1, wherein the Clostridium is Clostridium perfringes.10. The method of claim 1, wherein the Clostridium is Clostridiumdifficile.
 11. The method of claim 1, further comprising modifying theBacillus strains administered based on a change in Clostridium strainsto which the pig is exposed.
 12. The method of claim 1, whereinadministration of the Bacillus strains provides at least one of thefollowing in litters borne to the pig relative to that in litters borneto pigs that have not been administered the Bacillus strains: (A) loweraverage scour scores in the first week after birth, and (B) a decreasein the percentage of scouring litters.
 13. The method of claim 1,wherein the pig is in a herd lacking symptoms of Clostridium infection.14. The method of claim 13, wherein administration of the Bacillusstrains decreases clostridial load in the gastrointestinal tract ofpiglets borne by the pig relative to that in piglets borne to pigs thathave not been administered the Bacillus strains.
 15. The method of claim1, wherein the pig is in a unit of pigs that are subclinical forclostridial scours.
 16. The method of claim 15, wherein performance ofat least one of the pig and piglets borne by the pig is improved duringlactation relative to pigs and piglets that have not been administeredthe Bacillus strains.
 17. The method of claim 1, wherein the Bacillusstrain is administered when the pig is at least one of a nursery pig anda feedlot pig.
 18. A method comprising administering to a pig aneffective amount to inhibit Clostridium in litters borne to the pig ofBacillus strains 3AP4 (ATCC PTA-6506) and LSSAO1 (NRRL B-50104).
 19. Themethod of claim 18, wherein the pig is a gestating pig.
 20. The methodof claim 18, wherein the pig is a lactating pig.
 21. A method comprisingadministering to a pig an effective amount to inhibit Clostridium inlitters borne to the pig of at least two Bacillus strains having all ofthe identifying characteristics of at least two strains selected fromstrains 22CP1 (ATCC PTA-6508), 3AP4 (ATCC PTA-6506), 15AP4 (ATCCPTA-6507), 2084 (NRRL B-50013), LSSAO1 (NRRL B-50104), and 27 (NRRLB-50105), wherein at least one Bacillus strain has all of theidentifying characteristics of LSSAO1 (NRRL B-50104).
 22. The method ofclaim 21, wherein the Bacillus strains have all of the identifyingcharacteristics of strains 3AP4 (ATCC PTA-6506) and LSSAO1 (NRRLB-50104).
 23. The method of claim 21, wherein the Bacillus strains haveall of the identifying characteristics of strains 15AP4 (ATCC PTA-6507)and LSSA01 (NRRL B-50104).